Abstract
The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp. and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate (methyl-(butylcarbamoy 1)-2-benzimidazole carbamate), Tachigaren (3-hydroxy-5-methylisoxazole) and other chemicals were also clarified. The results are summarizee as follows: 1. There were two biologic strains, Type-l and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l being more virulent. 2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/l of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not. 3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while rnaltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results. 4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon Sources were applied but decreased with the increase of nitrogen above 0.5g/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for maximun growth of mycelia. At a higher concentration of more than 20r/l, however, mycelial growth decreased as the concentration increased, and was inhibited at l50r/l to such a degree of thiamine-free. 6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, and 16r/l in asparagine for the production of sclerotia. 7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2. 8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine+biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited. Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone. 9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ml/50ml basal medium solution. 10. $NH_4NO_3$. as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate. 11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed. and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction. 12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin. 13. Seven amino acids-aspartic acid, cystine, glysine, histidine, Iycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids. 14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied .alone and when combined with other amino acids in some cases. 15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ germinability, respectively. 17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l and type-2 were killed when dipped into the $0.05\%$ solution of mercury chloride for 180 mins. and 240 mins. respectively: and in the $0.1\%$ solution, Type-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the$0.125\%$solution. Dipping into the $5\%$ copper sulphate solution or $0.2\%$ solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-l or Type-2. 19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by $66\%$ at 0.5ppm and by $92\%$ at 2.0ppm of Benlate, and by$54\%$ at 1ppm and about $77\%$ at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlate ant at 500ppm or 1,000ppm of Tachigaren. 20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the ontrol. 21. The amount of mycelia produced by consuming 1mg of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time. 22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/g sand of Benlate or Tachigaren. 23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigaren for 10 minutes or even 20 minutes.
본 연구는 목련에서 분리한 흰비단병균 Sclerotimu rolfsii Sacc.의 분화형을 밝히고 균계생장 및 균핵형성에 대한 영양생리를 구명코저 vitamin, 질소원, 탄소원의 효과를 검토했으며 또 본 균과 Penicillium sp.와의 생태적 관계를 해명하기 위한 기초적인 연구로서 본 균의 균계생장 및 균핵형성에 대한 Penicillium 배양여액의 촉진효과와 그 요인을 밝히려고 시도하였다. 본 연구결과를 종합해서 적요하면 다음과 같다. 1. 목연에서 분리한 흰비단병균 제1형, 축2형은 배지상의 성상이나 생리적 성질 펄 병원성이 상이하였다. 특히 목연 아카시아에 대한 병원성은 양자 동일하나 콩이나 오이에 대해서는 제2형균이 제1형균보다 더 강하였다. 2. 공시된 14종의 질소원중 $KNO_2$와 glycine을 제외하고는 모두 thiamine hydrochloride 10r/l가 첨가되었을 때 비로소 공시균의 균계생장 및 균핵형성에 이용되었다. 질소의 형태별로 보면 균계생장에 있어서는 $NO_3-N$보다는 $NH_4-N$이 훨씬 더 효과적이며 organic N은 화합물에 따라 상이하였다. 그러나 균핵형성에 있어서는 이와 반대로 $NO_3-N$이 효과적이었다. $NO_2-N$은 균계생장이나, 균핵형성에 전혀 효과가 없었다. 3. 공시 탄소원 7종도 대체적으로 thiamine이 존재하지 않는 한 균계생장이나 균핵형성에 아무런 효과가 없었다. thiamine이 첨가될 경우 균계생장에 있어서 glucose와 saccharose가 가장 효과적이고 maltose와 soluble starch는 효과가 적었으며 xylose, lactose, glyceline은 전혀 효과가 없었다. 균핵형성에 있어서는 loctose를 제외하고는 전구에서 균핵형성을 보였으며 모두 비슷한 효과를 나타냈다. 4. 배지중의 질소원이 동일수준이면 탄소원이 증가함에 따라 균계생장량이 증가하였다. 그러나 질소원의 량에는 한도가 있는 것으로 질소 0.5g/l이상에서는 오히려 균계생장이 억제되었다. 그러나 균핵형성에 있어서는 탄소원의 증가에 따라 균핵형성량이 저하하였다. 5. 공시균은 thiamine 결핍균으로서 균계생장 최적 thiamine 농도는 20r/l이고 이 농도를 초과하면 오히려 균계생장이 억제되는데 150r/l에서는 무첨가구와 거의 같은 정도로 억제되었다. 6. 공시균의 생장에 있어서 thiamine의 첨가에 따른 질소원리용도는 $NH_4NO_3>(NH_4)_2SO_4>asparagine>KNO_3$의 순위이며 질소원별 thiamine 최적요구량은 $KNO_3$인 경우 12r/l, asparagine인 경우 16r/l 정도였다. 균핵형성량에 있어서는 $KNO_3>NH_4NO_3>asparagine>(NH_4)_2SO_4$의 순위로서 thiamine 최적량은 $KNO_3,\;NH_4NO_3$인 경우 8r/l에서 전균핵생산량의 대부분이 형성되나 asparagine인 경우에는 16r/l 정도였다. 7. 배양액의 pH는 공시균이 생장을 개시하자마자 3.5정도로 급격히 떨어지나 그 이후부터는 생장량이 증가함에 따라 완만하게 떨어졌다. 그러나 pH2.2 이하로는 더 내려가지 않았다. 8. 공시균의 균계생장에 대한 각종 vitamin의 상호효과는 thiamine, biotin, pyridoxine, inositol의 4가지 조합에 있어서도 thiamine이 첨가되지 않은 곳에서는 거의 효과가 나타나지 않았다. 그러나 thiamine+pyridoxine, thiamine+inosital, thiamine+biotin+pyridoxine, thiamine+pyridoxine+inositol구에 있어서는 thiamine 단독 첨가구와 동등 혹은 그 이상의 효과를 나타내지만 thiamine+biotin과 thiamine+biotin+inosital구는 오히려 떨어졌다. 균핵형성에 있어서는 thiamine 단독구에 비하여 각구 모두 약간씩 증가하였다. 9. Penicillium 배양여액중에는 공시균의 균계생장을 촉진하는 물질이 존재하며 배양여액 6-15ml/50ml 배양액의 농도에서 거의 최고균계생장량에 달하였다. 10. 질소원으로서 첨가한 $NH_4NO_3$ 혹은 asparagine은 균계생장에 있어서 배양여액농도 여가에 관계없이 $NH_4NO_3$가 더 유효하였다. 11. 배양여액에 대한 일련의 처리에 있어서 휘발성물질분획, 비휘발성물질분획, 휘발산분획, ether 가용성유기산분획, ether 불용성물질분획, cation 흡착물질분획, cation 비흡착물질분획, anion 흡착물질분획 및 비흡착물질분획의 9분획중 비휘발성물질분획, ether 불용물질분획, cation 흡착물질분획 및 anion 비흡착물질분획에서만 균계가 잘 자랐다. 그러나 균핵은 오직 cation 흡착물질분획에서만 형성되었다. 12. 이 결과는 배양여액중에 균계생장물질, 균핵형성물질 및 균핵형성억제물질이 존재하며 이들 물질은 각각 별개의 물질로서 전2자는 비휘발성이고 ether 불용성이며 cation교환수지에는 흡착되지 않는 물질이며 후자는 비휘발성이고 ether 불용성이며 cation 및 anion 교환수지에 흡착되지 않는 물질임을 암시한다. 13. DNP-aminoacids paper chromatography에 의하여 cation 교환수지흡착분획중에서 aspartic acid, cystine, glycine, histidine, lycine, tyrosine 및 dinitroaniline 7종의 아미노산이 검출되었다. 14. S. rolfsii의 균계생장 및 균핵형성은 glutamic acid, aspartic acid, cystine, histidine 및 glycine의 단독첨가나 혼합첨가에 의해서 촉진되지 않았고 다만 tyrosine에 의해서 약간 촉진되었다. 15. 균핵의 습열에 대한 저항성은 균핵의 수분함유량에 따라 다르며 수분함유량이 적은 것이 보다 더 강하였다. 배지에서 채취한 균핵은 제1,2형균 모두 $52^{\circ}C$에서 5분에 사멸하거나 155일간 $26{\circ}C$에서 건조시킨 것은 $52^{\circ}C$에 있어서 제1형균은 15분, 제2형균은 10분, $57^{\circ}C$에 있어서는 제1형균은 5분, 제2형균은 10분처리에 사멸하였다. 16. 배양균핵을 132일간 $26^{\circ}C$에서 건조시킨 것은 제1,2형균 모두 전부 발아하였고 기건상태에서 283일간 방치한 천연균핵도 제1,2균형 모두 발아하였으며 443일간처리한 것도 아직 제1형균 $20\%$, 제2형균 $16$의 발아율을 보지하고 있었다. 17. 저온에 대한 저항성은 균계, 균계괴, 균핵의 순으로 강하였는데 균계는 $-7--8^{\circ}C$ 1주간 처리에서 완전 사멸하였으나 균계괴는 $-17--20^{\circ}C$ 3주에서도 아직 사멸치 않은 것이 있었으며 균핵은 $-17--20^{\circ}C$ 3주에서 대부분 생존하였다. 18. 약제저항력은 승괴수 $0.05%$에 있어서 제1형균은 180분 제2형균은 240분, $0.1\%$에 있어서는 제1형균 60분, 제2형균 30분에 각각 사멸하였고, Uspulun 800배에 있어서는 제1형균은 120분에 사멸하나 제2형균은 180분에도 사멸치 않으며 500배에 있어서는 제1,2형균 모두 90분에 비로소 사멸하였다. 그러나 유산동 $5\%$ 240분, Ceresan 석탄, Mercuron 각 500배 80분처리에도 아무런 영향이 없었다. 19. Benlate와 Tachigaren의 농도가 증가함에 따라 균계생장 억제효과도 증가하였다. 처리 6째에는 Benlate 0.5ppm을 제외하고는 전농도에서 뚜렷한 억제효과를 나타내었으나 12일째에는 농도에 따라 현저한 차이를 나타냈다. Benlate 0.5ppm 처리는 대조구에 비하여 $66\%$, 2.0ppm은 $92\%$의 억제효과를, Tachigaren은 1ppm $54\%$, 1.5ppm과 2.0ppm은 $77\%$의 억제효과를 나타냈다. 양자 모두 500ppm에서는 거의 완전히 균계생장을 억제시켰다. 균핵형성은 Benlate 500ppm과 Tachigaren 500ppm 및 1000ppm에서 $100\%$ 억제되었다. 20. 일반적으로 균계생장량이 증가함에 따라 배지중의 glucose나 $NH_4-N$의 소비량도 증가하였으나 Benlate나 Techigaren을 처리할 경우 그 농도의 증가에 따라 이들의 소비량이 억제되었다. 그러나 Benlate 저농도(0.5ppm 및 1ppm)에 있어서는 $NH_4-N$의 소비가 무처리구보다 많았다. 21. glucose와 $NH_4-N$의 흡수이용효과 즉 glucose나 $NH_4-N$ 1mg을 소비하여 생산된 균계량은 Benlate나 Techigaren의 처리로 말미암아 크게 저하되었다. 그 정도는 농도에 관계없이 처리 3일째에 가장 심했고 이후 시일이 경과함에 따라 높아졌다. Benlate 처리의 glucose를 제외하고는 대체로 농도가 증가함에 따라 흡수이용효과가 저하되었다. 22. 토양배양에 있어서 $CO_2$ 배출량으로 측정한 균계생장은 어느 농도에서나 저지되지 않았고 다만 Tachigaren 100mg/g 토양에서만 현저하게 억제되었다. 균핵형성은 Benlate나 Tachigaren 10mg/g 토양에서 완전히 억제되었다. 23. Benlate와 Tachigaren 0.1, 1.0, 10, 100, 1000ppm에 10분 및 20분간 침지처리한 결과 균핵의 발아억제효과를 인정할 수 없었다.
